SYNCHRON® System(s)
Chemistry Information Sheet
AMPH
Amphetamines
REF 475000
For In Vitro Diagnostic Use
ANNUAL REVIEW
Reviewed by:
Date
Reviewed by:
Refer to coversheet in front of method
PRINCIPLE
INTENDED USE
®
®
AMPH reagent, when used in conjunction with SYNCHRON LX System(s), UniCel DxC 600/800
®
System(s) and SYNCHRON Systems Drugs of Abuse Testing (DAT) Urine Calibrators, is intended for
the qualitative determination of amphetamines in human urine, at a cutoff value of 1000 ng/mL.
The AMPH assay provides a rapid screening procedure for determining the presence of amphetamines
(AMPH) and its metabolites in urine. This test provides only a preliminary analytical result; a positive
result by this assay should be confirmed by another generally accepted non-immunological method such
as thin layer chromatography (TLC), gas chromatography (GC), or gas chromatography/mass
1,2
spectrometry (GC/MS). GC/MS is the preferred confirmatory method.
Clinical consideration and professional judgement should be applied to any drug of abuse test result,
particularly when preliminary positive results are used.
CLINICAL SIGNIFICANCE
Amphetamines are a class of central nervous system stimulants. The most common amphetamines
include d-amphetamine, d,l-amphetamine, and d-methamphetamine. Measurements of amphetamines on
®
the SYNCHRON System(s) are used in the diagnosis and treatment of amphetamine use and overdose,
and in monitoring the presence of amphetamine to ensure appropriate therapy.
METHODOLOGY
3
This assay utilizes a homogenous enzyme immunoassay method. The AMPH reagent is comprised of
specific antibodies which can detect amphetamine and/or methamphetamine in urine. A drug-labeled
glucose-6-phosphate dehydrogenase (G6PDH) conjugate competes with any free drug from the urine
sample for a fixed amount of antibody binding sites. In the absence of free drug from the sample, the
drug-labeled G6PDH conjugate is bound by the specific antibody and enzyme activity is inhibited. This
reaction creates a direct relationship between the presence of drug and enzyme activity. The G6PDH
enzyme activity is determined spectrophotometrically by measuring its ability to convert nicotinamide
adenine dinucleotide (NAD) to NADH (reduced form).
®
The SYNCHRON System(s) automatically proportions the appropriate sample and reagent volumes into
a cuvette. The ratio for AMPH is one part sample to 12.5 parts reagent. The system monitors the change
in absorbance at 340 nanometers to calculate and express a reaction rate. A qualitative result is reported
based on a comparison of the sample rate to the calibrated cutoff rate.
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CHEMICAL REACTION SCHEME
GENERAL DISCUSSION
Amphetamines are synthetic derivatives of ephedrine. Due to their mood elevating properties,
4
amphetamines are subject to widespread abuse, particularly in industrial societies. When amphetamine
is ingested, it is readily absorbed in the GI tract and effects persist for 4 - 24 hours. Amphetamines
appear in urine within about 3 hours following oral administration. Urinary excretion is pH-dependent and
5
is enhanced in acidic pH.
SPECIMEN
TYPE OF SPECIMEN
Freshly collected urine samples should be used for testing. Collect urine samples in glass or plastic (i.e.,
polypropylene, polycarbonate, polyethylene) containers. Urine samples should be collected in the manner
6
7
routinely used for drug screening analysis. Samples should be at room temperature for testing.
SPECIMEN STORAGE AND STABILITY
2,6
If the sample cannot be analyzed immediately, it may be stored at +2°C to +8°C for up to 7 days.
If
longer storage is required or when a split sample collection method is used, samples should be stored
6
frozen at -20°C or less.
ADDITIONAL SPECIMEN STORAGE AND STABILITY CONDITIONS AS DESIGNATED BY THIS LABORATORY:
Refer to “Sample Integrity in Chemistry” write up in “Policies and Procedures” manual
SAMPLE VOLUME
The optimum volume, when using a 0.5 mL sample cup, is 0.3 mL of sample. For optimum primary
sample tube volumes and minimum volumes, refer to the Primary Tube Sample Template for your
system.
CRITERIA FOR UNACCEPTABLE SPECIMENS
Refer to the PROCEDURAL NOTES section of this chemistry information sheet for information on
unacceptable specimens.
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CRITERIA FOR SAMPLE REJECTION AS DESIGNATED BY THIS LABORATORY:
Refer to “Sample Integrity in Chemistry” write up in “Policies and Procedures” manual
PATIENT PREPARATION
SPECIAL INSTRUCTIONS FOR PATIENT PREPARATION AS DESIGNATED BY THIS LABORATORY:
Refer to “Sample Integrity in Chemistry” write up in “Policies and Procedures” manual
SPECIMEN HANDLING
SPECIAL INSTRUCTIONS FOR SPECIMEN HANDLING AS DESIGNATED BY THIS LABORATORY:
Refer to “Sample Integrity in Chemistry” write up in “Policies and Procedures” manual
REAGENTS
CONTENTS
Each kit contains the following items:
One AMPH Reagent Cartridge (1 x 250 tests)
VOLUMES PER TEST
Sample Volume
20 µL
Total Reagent Volume
250 µL
Cartridge Volumes
A
200 µL
Antibody/Substrate Reagent
B
50 µL
Enzyme Conjugate Reagent
C
––
REACTIVE INGREDIENTS
REAGENT CONSTITUENTS
Antibody/Substrate Reagent
69 mL
Monoclonal anti-amphetamines antibodies (mouse)
Glucose-6-phosphate (G6P)
Nicotinamide adenine dinucleotide (NAD)
Tris buffer
Enzyme Conjugate Reagent
18 mL
Glucose-6-phosphate dehydrogenase (G6PDH) labeled with amphetamines
Tris buffer
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REAGENT CONSTITUENTS
Also non-reactive chemicals necessary for optimal system performance.
CAUTION
Sodium azide preservative may form explosive compounds in metal drain
lines. See National Institute for Occupational Safety and Health Bulletin:
Explosive Azide Hazards (8/16/76).
MATERIALS NEEDED BUT NOT SUPPLIED WITH REAGENT KIT
SYNCHRON Systems DAT Negative Urine Calibrator (0 ng/mL d-methamphetamine)
SYNCHRON Systems DAT Multi-Drug Low Urine Calibrator (1000 ng/mL d-methamphetamine)
SYNCHRON Systems DAT Multi-Drug High Urine Calibrator (2000 ng/mL d-methamphetamine)
SYNCHRON Systems DAT Multi-Drug Low Urine Control (750 ng/mL d-methamphetamine)
SYNCHRON Systems DAT Multi-Drug High Urine Control (1250 ng/mL d-methamphetamine)
REAGENT PREPARATION
No preparation is required.
ACCEPTABLE REAGENT PERFORMANCE
The acceptability of a reagent is determined by successful calibration and by ensuring that quality control
results are within acceptance criteria. Refer to the Quality Control section of this chemistry information
sheet for Substance Abuse and Mental Health Services Administration (SAMHSA) guidelines.
REAGENT STORAGE AND STABILITY
AMPH reagent, when stored unopened at +2°C to +8°C, will remain stable until the expiration date printed
on the cartridge label. Once opened, the reagent is stable for 90 days at +2°C to +8°C unless the
expiration date is exceeded. DO NOT FREEZE.
REAGENT STORAGE LOCATION:
Chemistry section, room L568. Refer to reagent “map” on Chemistry refrigerator #8.
CALIBRATION
CALIBRATOR REQUIRED
SYNCHRON Systems DAT Negative Urine Calibrator (0 ng/mL d-methamphetamine)
SYNCHRON Systems DAT Multi-Drug Low (cutoff) Urine Calibrator (1000 ng/mL d-methamphetamine)
SYNCHRON Systems DAT Multi-Drug High Urine Calibrator (2000 ng/mL d-methamphetamine)
CALIBRATOR PREPARATION
No preparation is required.
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CALIBRATOR STORAGE AND STABILITY
®
SYNCHRON Systems Drugs of Abuse Testing (DAT) Urine Calibrators are stable until the expiration
date printed on the calibrator bottles if stored capped in the original containers at +2°C to +8°C.
CAUTION
Urine is not known to transmit infectious disease such as Hepatitis or HIV.
However, because this product contains material of human origin, it should be
handled as though capable of transmitting infectious diseases. The United
States Food and Drug Administration recommends such samples be handled
as specified in the Centers for Disease Control`s Biosafety Level 2 guidelines.8
CALIBRATOR STORAGE LOCATION:
Chemistry section, room L568. Refer to reagent “map” on Chemistry refrigerator #8
CALIBRATION INFORMATION
1. The DAT assays require three levels of calibrators. The calibration measures the separation between
calibrators to ensure reagent integrity.
NOTICE
The calibration factor generated is non-functional for sample result calculation.
2. The system must have a valid calibrator cutoff value in memory before controls or patient samples can
be run. The cutoff value for each DAT chemistry represents the mean reaction rate of the Low
Calibrator, and is reported in mA/min units on patient and control reports. Cutoff values are stored in
memory until the next successful calibration.
3. Under typical operating conditions the AMPH reagent cartridge must be calibrated every 14 days and
also with certain parts replacements or maintenance procedures, as defined in the SYNCHRON LX
Maintenance Manual and Instrument Log, or the UniCel DxC 600/800 System Instructions For Use
(IFU) manual. This assay has within-lot calibration available. Refer to the SYNCHRON LX Operations
Manual, or the UniCel DxC 600/800 System Instructions For Use (IFU) manual for information on this
feature.
4. For detailed calibration instructions, refer to the SYNCHRON LX Operations Manual, or the UniCel
DxC 600/800 System Instructions For Use (IFU) manual.
5. The system will automatically perform checks on the calibration and produce data at the end of
calibration. In the event of a failed calibration, the data will be printed with error codes and the system
will alert the operator of the failure. For information on error codes, refer to the SYNCHRON LX
Diagnostics and Troubleshooting Manual, or the UniCel DxC 600/800 System Instructions For Use
(IFU) manual.
TRACEABILITY
For Traceability information refer to the Calibrator instructions for use.
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QUALITY CONTROL
Good laboratory practices suggest the use of control specimens to ensure proper assay performance.
Each analytical run should include controls with levels 25% above and 25% below the cutoff threshold of
9
each drug, as well as negative specimens certified to contain no drug. In addition, these controls should
be run with each new calibration, and after specific maintenance or troubleshooting procedures as
detailed in the appropriate system manual. More frequent use of controls or the use of additional controls
is left to the discretion of the user based on good laboratory practices or laboratory accreditation
requirements and applicable laws.
The following controls should be prepared and used in accordance with the package inserts. Discrepant
quality control results should be evaluated by your facility.
TABLE 1 QUALITY CONTROL MATERIAL
CONTROL NAME
SAMPLE TYPE
STORAGE
Bio-Rad S10 and S20 vials in use kept refrigerated after thawing. Unopened S10 and S20 kept frozen until
just before use. Refer to “DXC 800 Control Analysis” in DXC 800 procedure manual for other control material
used and storage. Control preparations and acceptance of QC results are in “Policies and Procedures”
manual
TESTING PROCEDURE(S)
1. If necessary, load the reagent onto the system.
2. After reagent load is completed, calibration may be required.
3. Program samples and controls for analysis.
4. After loading samples and controls onto the system, follow the protocols for system operations.
For detailed testing procedures, refer to the SYNCHRON LX Operations Manual, or the UniCel DxC
600/800 System Instructions For Use (IFU) manual.
RESULTS INTERPRETATION
The system performs all calculations internally to produce the final qualitative result, reported as
POSITIVE or NEGATIVE. The qualitative result is based on a comparison of the sample rate to the
calibrated cutoff rate; a sample rate greater than or equal to the cutoff rate is reported as POSITIVE. A
POSITIVE result (≥1000 ng/mL) from this assay indicates only the presence of this analyte and does not
necessarily correlate with the extent of physiological and psychological effects. A NEGATIVE test result
indicates that this analyte is either not present, or is present at levels below the cutoff threshold of the
test.
REPORTING RESULTS
Equivalency between the SYNCHRON LX and UniCel DxC 600/800 Systems has been established.
Chemistry results between these systems are in agreement and data from representative systems may
be shown.
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ADDITIONAL REPORTING INFORMATION AS DESIGNATED BY THIS LABORATORY:
Refer to “DXC800 Linearity and Reportable Range” chart in Technical notes section of DXC800 Procedure
manual
PROCEDURAL NOTES
LIMITATIONS
1. The test is designed for use with human urine only.
2. Do not dilute the urine samples since this is a qualitative assay. Dilution of samples may produce
erroneous results.
3. Interference has been demonstrated from mefenamic acid, a nonopiod analgesic (which absorbs at
10
340 nm).
4. Adulteration of the urine sample may cause erroneous results. Alteration of a urine specimen may be
detected by checking the appearance, temperature, pH specific gravity, and creatinine levels of a
6
sample. If adulteration is suspected, obtain another sample and forward both specimens to the
laboratory for testing.
5. An effort should be made to keep pipetted samples free from gross debris. It is recommended that
highly turbid specimens be centrifuged before analysis.
6. Interference may be caused by other substances and/or factors (e.g., technical or procedural errors)
not listed above, producing false results.
Note: For screening assays of this type, only POS or NEG is reported. Occasionally error codes, e.g.
Reaction Rate Hi/Low, Blank Rate Hi/Low,Reaction ABS Hi/Low may be sent from the DXC. The error
messages may be due to sample integrity or a possible interfering substance. Rarely, the error may be
due to bubbles in the reagent. Whenever an error code is generated, repeat the test. If the repeat assay
generates the same DXC error code, in Sunquest send English Text code INTC “Interfering Substance,
unable to do” as the result. Do not dilute the sample.
PERFORMANCE CHARACTERISTICS
RELATIVE SENSITIVITY AND SPECIFICITY
One hundred twelve clinical urine specimens were collected and tested. 91% agreement was obtained
between AMPH Reagent P/N 445965 (current) and improved AMPH Reagent P/N 475000 (new).
Six samples tested negative with the new reagent. Four samples, shown by GC/MS to contain ranitidine
but no amphetamines, correctly tested negative with the new reagent, whereas they previously tested
false positive. Two samples testing negative with the new reagent, and positive with the current reagent,
had rates within 0.8% and 1.3% of the cutoff rate (284 mA/min).
Four samples tested negative with the current reagent and positive with the new reagent. Two of the four
had borderline rates (within 3.5% of the cutoff rate), and two contained phenylpropanolamine,
pseudoephedrine and ephedrine.
TABLE 2 CURRENT REAGENT VS. NEW REAGENT
AMPH
REAGENT
AMPH
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P/N
Positive
Positive
44
11
REAGENT P/N 475000
Negative
6
Total
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AMPH
445965
Negative
Total
Positive
4
REAGENT P/N 475000
Negative
58
Total
62
48
64
112
Relative Sensitivity (% agreement among positives): 88%
Relative Specificity (% agreement among negatives): 94%
Overall Agreement: 91%
Forty-one positive samples were analyzed by GC/MS. Twenty-three samples were confirmed positive by
GC/MS. Sixteen samples gave negative GC/MS results. Analysis of these samples showed the presence
of phenylpropanolamine, ephedrine, and pseudoephedrine. These compounds are known potential cross
reactants and are listed in Table 3.0 Cross Reactivity. One sample contained methadone and ranitidine
and one sample contained amphetamine and methamphetamine at levels slightly below the GC/MS cutoff
of 1000 ng/mL.
CROSS REACTIVITY
Amphetamines, methamphetamines, amphetamine-like compounds, and various potential interfering
substances in a human urine matrix were tested for cross-reactivity with the SYNCHRON Systems AMPH
assay. The following table summarizes the results obtained at the concentrations listed for each potential
cross-reactant.
Table 3 Cross Reactivity
a
COMPOUND
CONCENTRATION (µg/mL)
1
EFFECT
Positive
1
Positive
Methylenedioxyamphetamine (MDA)
2.5
Positive
Methylenedioxymethamphetamine (MDMA)
2.5
Positive
Acetaminophen
1000
Negative
Acetylsalicylic Acid
1000
Negative
l-Amphetamine
12.5
Negative
Benzoylecgonine
1000
Negative
Benzphetamine
20
Negative
Bupropion
50
Negative
Buspirone
1000
Negative
Caffeine
1000
Negative
Chlorpromazine
500
Negative
Codeine
1000
Negative
Dextromethorphan
1000
Negative
d-Ephedrine
400
Negative
d,l-Ephedrine
500
Negative
l-Ephedrine
350
Negative
4
Negative
3-Hydroxy-Tyramine
500
Negative
Isoxsuprine
100
Negative
d-Methamphetamine (cutoff)
d-Amphetamine
Fenfluramine
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COMPOUND
CONCENTRATION (µg/mL)
1000
EFFECT
Negative
25
Negative
1000
Negative
l-Methamphetamine
10
Negative
Methapyrilene
500
Negative
Morphine
1000
Negative
Nor-pseudoephedrine
1000
Negative
Oxazepam
500
Negative
Phencyclidine
1000
Negative
Phendimetrazine
200
Negative
Phenethylamine
10
Negative
Phenmetrazine
50
Negative
Phenobarbital
1000
Negative
Phenothiazine
10
Negative
Phentermine
25
Negative
Phenylephrine
500
Negative
Phenylpropanolamine (PPA)
250
Negative
Procainamide
20
Negative
Promethazine
500
Negative
Propranolol
200
Negative
d-Pseudoephedrine
250
Negative
l-Pseudoephedrine
3000
Negative
Ranitidine
1000
Negative
Scopolamine
100
Negative
Secobarbital
1000
Negative
Setraline
1000
Negative
Thioridazine
1000
Negative
Trifluoperazine
1000
Negative
Triflupromazine
1000
Negative
Trazodone
1000
Negative
Tyramine
500
Negative
Meperidine
Mephentermine
Methadone
PRECISION
The following estimates of within-run imprecision were obtained when 20 replicates of the Negative
Calibrator, Control 1 (750 ng/mL), Calibrator 1 (1000 ng/mL), Control 2 (1250 ng/mL), and Calibrator 2
(2000 ng/mL) were assayed on a properly operated and maintained SYNCHRON LX System.
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TABLE 4 TYPICAL WITHIN-RUN IMPRECISION
SAMPLE
MEAN RATE (mA/min)
185
1 SD (mA/min)
0.9
% CV
0.5
Control 1
271
1.7
0.6
Cal 1
291
1.5
0.5
Control 2
308
1.7
0.5
Cal 2
330
2.0
0.6
Negative Cal
All of the negative calibrator and low control rates were negative. All of the high calibrator and high control
rates were positive. The low (cutoff) calibrator rates were uniformly distributed around the cutoff rate.
Each laboratory should characterize their own instrument performance for comparison purposes.
Instruments operated and maintained according to the manufacturer`s instructions should exhibit a withinrun coefficient of variation of ≤ 2.0% for all sample levels.
NOTICE
These degrees of precision and equivalency were obtained in typical testing procedures on a
®
SYNCHRON LX System and are not intended to represent the performance specifications for
this reagent.
ADDITIONAL INFORMATION
For more detailed information on SYNCHRON LX Systems or UniCel DxC Systems, refer to the
appropriate system manual.
SHIPPING DAMAGE
If damaged product is received, notify your Beckman Coulter Clinical Support Center.
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REFERENCES
1. National Institute on Drug Abuse Research, "Urine Testing for Drugs of Abuse", Monograph 73
(1986).
2. National Institute on Drug Abuse, "Mandatory Guidelines for Federal Workplace Drug Testing
Programs", Federal Register, Vol. 53, No. 69 (1988).
3. Rubenstein, K. E., Schneider, R. S., Ullman, E. F., 'Homogenous Enzyme Immunoassay: A New
Immunochemical Technique", Biochem. Biophys. Res. Commun. , Vol. 47, 846 851 (1972).
4. McEnvoy, G. K., Litvak, K., AHFS Drug Information, American Society of Hospital Pharmacists,
Inc., Bethesda, MD (1992).
5. Wyngarrden, J. B., Smith, L. H. Jr., Cecil, Textbook of Medicine, W. B. Saunders Company,
Philadelphia, PA (1982).
6. National Committee for Clinical Laboratory Standards, Urine Drug Testing in the Clinical
Laboratory, Proposed Guideline, Vol.13, No. 7, NCCLS publication T/DM8-P, Villanova, PA
(1993).
7. "USP XXII, NF XVII", United States Pharmacopeial Convention, Inc., Rockville, MD (1990).
8. CDC-NIH manual, Biosafety in Microbiological and Biomedical Laboratories, U.S. Government
Printing Office, Washington, D.C. (1984).
9. Substance Abuse and Mental Health Service Administration, "Mandatory Guidelines for Federal
Workplace Drug Testing Programs", Federal Register, Vol. 58, No. 14, (1993).
10. Crane, T., et al., "Mefenamic Acid Prevents Assessment of Drug Abuse with EMIT
Clin. Chem., Vol. 39, No. 3, 549 (1993).
™
Assays",
11. Tietz, N. W., Fundamentals of Clinical Chemistry, 4th Edition, W. B. Saunders Company,
Philadelphia, PA (1996).
Beckman Coulter Ireland Inc., Mervue Business Park, Mervue, Galway, Ireland
(353 91 774068)
Beckman Coulter, Inc., 250 South Kraemer Blvd., Brea, CA 92821
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ENDNOTES
a
It is possible that other substances and/or factors (e.g. technical or procedural) not listed above may
interfere with the test and cause false results. Data shown was collected using SYNCHRON CX
Systems. Equivalency between SYNCHRON LX Systems has been established by Deming
regression analysis to SYNCHRON CX Systems.
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